The present invention is related to novel nucleic acids and polypeptides encoding the catalytic subunit of telomerase. In particular, the present invention is directed to the catalytic subunit of telomerases from Euplotes aediculatus, Schizosaccharomyces pombe, Tetrahymena thermophila, and humans. The invention provides methods and compositions relating to medicine, molecular biology, chemistry, pharmacology, and medical diagnostic and prognostic technology.
The following discussion is intended to introduce the field of the present invention to the reader. The citation of various references in this section is not to be construed as an admission of prior invention.
It has long been recognized that complete replication of the ends of eukaryotic chromosomes requires specialized cell components (Watson, 1972, Nature NewBiol., 239:197; Olovnikov, 1973, J. Theor. Biol., 41:181). Replication of a linear DNA strand by conventional DNA polymerases requires an RNA primer, and can proceed only 5xe2x80x2 to 3xe2x80x2. When the RNA bound at the extreme 5xe2x80x2 ends of eukaryotic chromosomal DNA strands is removed, a gap is introduced, leading to a progressive shortening of daughter strands with each round of replication. This shortening of telomeres, the protein-DNA structures physically located on the ends of chromosomes, is thought to account for the phenomenon of cellular senescence (cell aging) of normal human somatic cells in vitro (see, e.g., Goldstein, 1990, Science 249:1129) and in vivo (see, e.g., Martin et al., 1979, Lab. Invest. 23:86; Goldstein et al., 1969, Proc. Natl. Acad. Sci. USA 64:155; and Schneider and Mitsui, 1976, Proc. Natl. Acad. Sci. USA, 73:3584).
The length and integrity of telomeres is thus related to entry of a cell into a senescent stage (i.e., loss of proliferative capacity). Moreover, the ability of a cell to maintain (or increase) telomere length may allow a cell to escape senescence, i.e., to become immortal.
The structure of telomeres and telomeric DNA has been investigated in numerous systems (see, e.g, Harley and Villeponteau, 1995, Curr. Opin. Genet. Dev. 5:249). In most organisms, telomeric DNA consists of a tandem array of very simple sequences; in humans and other vertebrates telomeric DNA consists of hundreds to thousands of tandem repeats of the sequence TTAGGG. Methods for determining and modulating telomere length in cells are described in PCT Publications WO 95/13382 and WO 96/41016.
The maintenance of telomeres is a function of a telomere-specific DNA polymerase known as telomerase. Telomerase is a ribonucleoprotein (RNP) that uses a portion of its RNA moiety as a template for telomere repeat DNA synthesis (Morin, 1997, Eur. J. Cancer 33:750; Yu et al., 1990, Nature 344:126; Singer and Gottschling, 1994, Science 266:404; Autexier and Greider, 1994, Genes Develop., 8:563; Gilley et al., 1995, Genes Develop., 9:2214; McEachern and Blackburn, 1995, Nature 367:403; Blackburn, 1992, Ann. Rev. Biochem., 61:113;. Greider, 1996, Ann. Rev. Biochem., 65:337). The RNA components of human and other telomerases have been cloned and characterized (see, PCT Publication WO 96/01835 and Feng et al., 1995, Science 269:1236). However, the characterization of the protein components of telomerase has been difficult. In part, this is because it has proved difficult to purify the telomerase RNP, which is present in extremely low levels in cells in which it is expressed. For example, it has been estimated that human cells known to express high levels of telomerase activity may have only about one hundred molecules of the enzyme per cell.
Consistent with the relationship of telomeres and telomerase with the proliferative capacity of a cell (i.e., the ability of the cell to divide indefinitely), telomerase activity is detected in immortal cell lines and an extraordinarily diverse set of tumor tissues, but is not detected (i.e., was absent or below the assay threshold) in normal somatic cell cultures or normal tissues adjacent to a tumor (see, U.S. Pat. Nos. 5,629,154; 5,489,508; 5,648,215; and 5,639,613; see also, Morin, 1989, Cell 59: 521; Shay and Bacchetti 1997, Eur. J. Cancer 33:787; Kim et al., 1994, Science 266:2011; Counter et al., 1992, EMBO J. 11:1921; Counter et al., 1994, Proc. Natl. Acad Sci. U.S.A. 91, 2900; Counter et al., 1994, J. Virol. 68:3410). Moreover, a correlation between the level of telomerase activity in a tumor and the likely clinical outcome of the patient has been reported (e.g., U.S. Pat. No. 5,639,613, supra; Langford et al., 1997, Hum. Pathol. 28:416). Telomerase activity has also been detected in human germ cells, proliferating stem or progenitor cells, and activated lymphocytes. In somatic stem or progenitor cells, and in activated lymphocytes, telomerase activity is typically either very low or only transiently expressed (see, Chiu et al., 1996, Stem Cells 14:239; Bodnar et al., 1996, Exp. Cell Res. 228:58; Taylor et al., 1996, J. Invest. Dermatology 106: 759).
Human telomerase is an ideal target for diagnosing and treating human diseases relating to cellular proliferation and senescence, such as cancer. Methods for diagnosing and treating cancer and other telomerase-related diseases in humans are described in U.S. Pat. Nos. 5,489,508, 5,639,613, and 5,645,986. Methods for predicting tumor progression by monitoring telomerase are described in U.S. Pat. No. 5,639,613. The discovery and characterization of the catalytic protein subunit of human telomerase would provide additional useful assays for telomerase and for disease diagnosis and therapy. Moreover, cloning and determination of the primary sequence of the catalytic protein subunit would allow more effective therapies for human cancers and other diseases related to cell proliferative capacity and senescence.
The present invention provides an isolated, substantially pure, or recombinant protein preparation of a telomerase reverse transcriptase protein. In one embodiment the protein has an amino acid sequence (SEQ ID NOS: 11-12):
Trp-R1-X7-R1-R1-R2-X-Phe-Phe-Tyr-X-Thr-Glu-X8-9-R3-R3-Arg-R4-X2-Trp
where X is any amino acid and a subscript refers to the number of consecutive residues, R1 is leucine or isoleucine, R2 is glutamine or arginine, R3 is phenylalanine or tyrosine, and R4 is lysine or histidine. In one embodiment the protein has a sequence of human TRT. In another embodiment, the invention relates to peptides and polypeptides sharing substantial sequence identity with a subsequence of such proteins.
In a related embodiment the invention provides an isolated, substantially pure or recombinant nucleic acid that encodes a telomerase reverse transcriptase protein. In one embodiment the protein has an amino acid sequence (SEQ ID NO: 11-12):
Trp-R1-X7-R1-R1-R2-X-Phe-Phe-Tyr-X-Thr-Glu-X8-9-R3-R3-Arg-R4-X2-Trp. In one embodiment the nucleic acid has a sequence of human TRT. In other embodiment, the invention relates to oligonucleotides and polynucleotides sharing substantial sequence identity with a subsequence of such nucleic acids.
In one aspect the invention provides isolated human telomerase comprising human telomerase reverse transcriptase (hTRT). In one embodiment the hTRT is associated with human telomerase RNA (hTR).
In one aspect the invention provides a method of detecting a human telomerase reverse transcriptase (hTRT) gene product in a biological sample by contacting the biological sample with a probe that specifically binds the gene product, wherein the probe and the gene product form a complex, and detecting the complex where the presence of the complex is correlated with the presence of the hTRT gene product in the biological sample. The gene product may be RNA, DNA or a polypeptide. Examples of probes in that may be used for detection include, but are not limited to, nucleic acids and antibodies.
In one embodiment the gene product is a nucleic acid which is detected by amplifying the gene and detecting the amplification product, where the presence of the complex or amplification product is correlated with the presence of the hTRT gene product in the biological sample.
In one embodiment the biological sample is from a patient, such as a human patient. In another embodiment the biological sample includes at least one cell from an in vitro cell culture, such as a human cell culture.
The invention further provides a method of detecting the presence of at least one immortal or telomerase positive human cell in a biological sample comprising human cells by obtaining the biological sample comprising human cells; and detecting the presence in the sample of a cell having a high level of an hTRT gene product, where the presence of a cell having a high level of the hTRT gene product is correlated with the presence of immortal or telomerase positive cells in the biological sample.
The invention also provides a method for diagnosing a telomerase-related condition in a patient by obtaining a cell or tissue sample from the patient, determining the amount of a human telomerase reverse transcriptase (hTRT) gene product in the cell or tissue; and comparing the amount of hTRT gene product in the cell or tissue with the amount in a healthy cell or tissue of the same type, where a different amount of hTRT gene product in the sample from the patient and the healthy cell or tissue is diagnostic of a telomerase-related condition. In one embodiment the telomerase-related condition is cancer.
The invention further provides a method of diagnosing cancer in a patient by obtaining a biological sample from the patient, and detecting a human telomerase reverse transcriptase (hTRT) gene product in the patient sample, where the detection of the hTRT gene product in the sample is correlated with a diagnosis of cancer.
The invention further provides a method of diagnosing cancer in a patient by obtaining a patient sample, determining the amount of human telomerase reverse transcriptase (hTRT) gene product in the patient sample; and comparing the amount of hTRT gene product with a normal or control value, where an amount of the hTRT gene product in the patient that is greater than the normal or control value is diagnostic of cancer.
The invention still further provides a method of diagnosing cancer in a patient, by obtaining a patient sample containing at least one cell; determining the amount of an hTRT gene product in a cell in the sample; and comparing the amount of hTRT gene product in the cell with a normal value for the cell, wherein an amount of the hTRT gene product greater than the normal value is diagnostic of cancer. In one embodiment the sample is believed to contain at least one malignant cell.
The invention still further provides a method of providing a prognosis for a cancer patient by determining the amount of hTRT gene product in a cancer cell obtained from the patient; and comparing the amount of hTRT in the cancer cell with a prognostic value of hTRT per cancer cell consistent with a prognosis for the cancer; where an amount of hTR per cell in the sample that is at the prognostic value provides the particular prognosis.
The invention still further provides a method for monitoring the ability of an anticancer treatment to reduce the proliferative capacity of cancer cells in a patient, by making a first measurement of the amount of an hTRT gene product in at least one cancer cell from the patient; making a second measurement of the level of the hTRT gene product in at least one cancer cell from the patient, wherein the anticancer treatment is administered to the patient before or at the same time as the second measurement; and comparing the first and second measurements, where a lower level of the hTRT gene product in the second measurement is correlated with the ability of an anticancer treatment to reduce the proliferative capacity of cancer cells in the patient.
The invention also provides kits for the detection of a hTRT gene or gene product. In one embodiment the kit includes a container including a molecule selected from an hTRT nucleic acid or subsequence thereof, an hTRT polypeptide or subsequence thereof, and an anti-hTRT antibody.
The invention also provides methods of treating human diseases. In one aspect the invention provides a method for increasing the proliferative capacity of a vertebrate cell, such as a mammalian cell, by introducing a recombinant polynucleotide into the cell, wherein said polynucleotide comprises a sequence encoding a human telomerase reverse transcriptase (hTRT) polypeptide. In one embodiment the hTRT polypeptide has a sequence of SEQ ID NO. 2. In one embodiment the sequence is operably linked to a promoter. In one embodiment the hTRT has telomerase catalytic activity. In one embodiment the cell is human, such as a cell in a human patient. In an alternative embodiment, the cell is cultured in vitro. In a related embodiment the cell is introduced into a human patient.
The invention further provides a method for treating a human disease by introducing recombinant hTRT polynucleotide into at least one cell in a patient. In one embodiment a gene therapy vector is used. In a related embodiment, the method further consists of introducing into the cell a polynucleotide comprising a sequence encoding human telomerase RNA, for example an hTR polynucleotide operably linked to a promoter.
The invention also provides a method for increasing the proliferative capacity of a vertebrate cell, said method comprising introducing into the cell an effective amount of a human telomerase reverse transcriptase (hTRT) polypeptide. In one embodiment the the hTRT polypeptide has telomerase catalytic activity. The invention further provides cells and cell progeny with increased proliferative capacity.
The invention also provides pharmacological compositions containing a pharmaceutically acceptable carrier and a molecule selected from: an hTRT polypeptide, a polynucleotide encoding an hTRT polypeptide, and an hTRT nucleic acid or subsequence thereof.
The invention also provides a method for treatment of a condition associated with an elevated level of telomerase activity within a cell, comprising introducing into said cell a therapeutically effective amount of an inhibitor of said telomerase activity, wherein said inhibitor is an hTRT polypeptide or an hTRT polynucleotide. In one embodiment the inhibitor is a polypeptide comprising the sequence of SEQ ID NO: 2 or 4, or a subsequence thereof. In additional embodiments the polypeptide inhibits a TRT activity, such as binding of endogenous TRT to telomerase RNA.
The invention also provides a vaccine comprising an hTRT polypeptide and an adjuvant.